Abstract How enhancers and promoters communicate is one of the important questions in the field of eukaryotic gene regulation. Despite provocative models from textbooks and review articles, very little is genuinely known of the mechanism. We were the first lab to show a direct interaction between TFIID and Mediator co-activators, and demonstrate that this interaction was required for Pol II preinitiation complex assembly in vitro. Based on these studies, we hypothesize that the key interaction driving Pol II preinitiation complex assembly and function is activator-regulated interaction between Mediator at the enhancer and TFIID at the promoter. We will leverage the knowledge and biochemical/genomewide technologies acquired from studies over the last three funding cycles to comprehensively identify proteins forming the TFIID-Mediator interface in vitro and determine if these interactions are central to gene expression and promoter-enhancer looping in cells. Our studies will employ biochemical and biological assays developed to analyze how the murine embryonic stem cell activator Esrrb activates transcription in vivo in murine embryonic stem cells (mESCs) and in vitro in extracts. This system will serve as a model and focal point for understanding the TFIID-Mediator interface. In Aim 1, we will identify the interface between Esrrb, Mediator and TFIID using crosslinking mass spectrometry (XL-MS). We will validate and further study these surfaces using protein-protein interaction assays and mutagenesis. In Aim 2, we will identify the TFIID-Mediator co-activator surface in vivo by systematic RNAi knockdown of co-activator subunits, individually and then in pairwise combinations, followed by RNA-seq to identify overlapping effects. We will perform genomewide ChIP-seq of TFIID and Mediator under mock and knockdown conditions to probe effects and correlate with the results from Aim 1. Aim 3 examines whether the TFIID-Mediator interface is necessary or not for promoter-enhancer looping in vivo using 4C. Our results will provide a detailed mechanism of eukaryotic gene activation using powerful state-of-the-art biochemical and biological approaches.